The primary objective of this proposal is to investigate the molecular mechanisms that underlie a genetically defined, natural transplantation reaction that occurs in the primitive chordate, Botryllus schlosseri. If two individuals of the same species share one or both alleles at the fuhc locus they will fuse together and form a chimera for the duration of the colonies lifespan. However, if no alleles are shared, the two colonies will reject in a blood-borne inflammatory reaction, preventing vascular fusion. In contrast to allo-responses, fuhcbased xenorecognition is not common: if B. schlosseri encounters a congener, the two species will grow into and over each other and the allorecognition reaction is never initiated. These observations suggest the presence of a species-level activation phase of the allorecognition response, followed by allelic discrimination of the fuhc. This type of recognition is reminiscent of the "missing-self recognition reactions utilized by the natural killer (NK) cells of the vertebrate innate immune system, which involve both activating and inhibitory steps during effector function. We have recently published the characterization of two proteins known to be involved in this reaction;the fuhc and its putative receptor, fester. Encoded between the two proteins is a gene with significant homology to fester, but is monomorphic and is being called uncle fester. This grant is focused on addressing the following questions: 1) what is the role of uncle fester? and 2) does uncle fester directly bind to the fuhc or fester? and 3) does uncle fester heterodimerize with fester or a subset of fester alternative splice variants? Experimentally, we will address these questions by: 1) characterizing uncle fester expression and function;2) performing direct binding assays between the three proteins and 3) by using an ex vivo assay to assess co-localization of uncle fester and fester. These studies are relevant for two reasons. First, as a primitive chordate, Botryllus occupies a key phylogenetic position within the chordate lineage and may reveal the origins of different components of vertebrate immunity. Second, Leukemia occurs when a group of blood cells begin to divide uncontrollably. Transplanting blood-forming stem cells from a healthy person could potentially replace the malignant cells, however, the host immune system usually rejects these bone-marrow transplants. We are studying a simple model organism which undergoes a natural transplantation reaction that closely resembles the rejection of a bone marrow transplant, and have recently identified some of the molecules involved. This allows us to study transplantation in a much simpler organism, allowing new insights into these processes.